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Neuron Review

How We Feel: Ion Channel Partnerships that Detect Mechanical Inputs and Give Rise to Touch and

Shana L. Geffeney1,2 and Miriam B. Goodman1,* 1Department of Molecular and Cellular , Stanford University, Stanford, CA 94305, USA 2Present address: Department of , Utah State University-Uintah Basin, 320 N. 2000 W. (Aggie Boulevard), Vernal, UT 84078, USA *Correspondence: [email protected] DOI 10.1016/j.neuron.2012.04.023

Every moment of every day, our and its embedded sensory are bombarded with mechanical cues that we experience as pleasant or painful. Knowing the difference between innocuous and noxious mechanical stimuli is critical for survival and relies on the function of neurons that vary in their size, shape, and sensitivity. Their function is poorly understood at the molecular level. This review emphasizes the importance of integrating analysis at the molecular and cellular levels and focuses on the discovery of ion channel proteins coexpressed in the of worms, flies, and mice.

Introduction activate or inhibit mechanoreceptor neurons can exert their influ- All sensory neurons are alike. Each detects a physical ence by acting on channels other than transduction channels. For and produces an electrical signal that gives rise to behavioral example, naked mole rats are insensitive to the persistent skin responses, conscious , or both. Many operate near acidification that is a feature of their environment. These animals the physical limits of detection and operate over a dynamic range have acid-gated ion channels (ASICs) with a similar sensitivity to of several orders of magnitude (Bialek, 1987; Block, 1992). These protons (H+) as those found in mice. However, the voltage-gated properties suggest that they are endowed with a detector, an Na+ channels expressed in their C-fiber are hyper- amplifier, and mechanisms for gain control. One of the most sensitive to inhibition by protons and this inhibition counterbal- striking and well-understood examples is the ability of photore- ances the excitation due to ASIC activation, rendering animals ceptors to detect single photons while retaining sensitivity to light insensitive to acidification (Smith et al., 2011). Thus, the differ- intensities that vary by nine orders of magnitude (Rieke and ence in sensitivity stems from variation in voltage- Rudd, 2009). gated Na+ sodium channels that are essential for Each somatosensory neuron is distinct. The somatosensory generation rather than any variation in sensory transduction. system is a collection of neurons innervating the skin, muscle, Though mechanoreceptor neurons were first studied more joints, , and internal organs that establish and maintain than 75 years ago (Adrian, 1926; Adrian and Zotterman, 1926a, sensitivity across a range of stimulus intensities and frequencies. 1926b), the events that link sensory stimulation to neuronal acti- This collection includes nociceptors that require stimulation vation are only beginning to be understood. Today, the protein above a high threshold for activation. Nociceptors are respon- partners responsible for detecting mechanical stimuli have sible for informing the about damage to peripheral tissues, been identified only for a few mechanoreceptor neurons in the an essential function that enables the nervous system to execute Caenorhabditis elegans. Genetic screens for animals and coordinate appropriate protective behaviors. They are often defective in touch sensation have revealed critical roles for polymodal neurons responding to multiple types of stimulation genes encoding TRP channels and DEG/ENaCs in behavioral including extreme , intense force, acid, and responses to mechanical inputs. The key insights derived from noxious chemicals. Other somatosensory neurons respond to genetic approaches have been reviewed elsewhere (Arnado´ ttir less intense stimulation and detect either changes and Chalfie, 2010; Ernstrom and Chalfie, 2002). We review or mechanical stimulation, but not both. These cells provide data demonstrating that TRP channels and DEG/ENaC channels information about warmth, cooling, or the shape and texture of are widely distributed in the sensory neurons of vertebrates and objects. invertebrates and examine the idea that these channels have conserved, but distinct functions. We rely on investigations of Theater of Sensation somatosensory mechanoreceptors in , flies, and The skin is our largest sensory surface, extending nearly two mice, but recognize that on-going investigations in square meters in an average . Mechanoreceptor neurons and other animals have the potential to deepen and expand are principal actors in this theater. They are responsible not only understanding of how mechanoreceptors function. for detecting mechanical cues, but also for encoding and trans- mitting all relevant information to the . Sensing with Ion Channels Their performance is shaped by ion channels that include, but For several decades, attention has focused on the idea that are not limited to, sensory transduction channels. Agents that mechano-electrical transduction (MeT) channels are formed by

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assemble into homomeric or heteromeric ion channels (Venkata- chalam and Montell, 2007). Recently, two additional classes of membrane proteins (Piezo and TMC) have been linked to mecha- notransduction (Coste et al., 2010; Coste et al., 2012; Kawa- shima et al., 2011; Kim et al., 2012). Both TRPs and DEG/ENaCs are broadly expressed in somato- sensory neurons. Several mechanoreceptor neurons are known to coexpress multiple TRPs and multiple DEG/ENaC channels. Figure 2 aggregates evidence that these channels are coex- pressed in mechanoreceptor neurons from the growing but essentially independent literatures on TRP and DEG/ENaC chan- nels expression and function. Excluding reviews, PubMed listed 1,687 entries for DEG/ENaCs, 2,341 for TRP channels, and only 15 entries for both ion channel families on April 15, 2012 (search conducted with the following search terms: ‘‘TRP channels,’’ ‘‘ENaC OR ASIC OR degenerin,’’ and the union of both terms). Here, we focus on two invertebrates, Caenorhabditis elegans nematodes and fruitflies, and one , the laboratory mouse. Despite the fact that members of these gene families are coexpressed, the function of individual TRP and DEG/ENaC channels is often explored subunit by subunit. But, genetic redundancy within each ion channel family and the potential for functional redundancy between the two Figure 1. Topology and Stoichiometry of Proteins Proposed to Form families limits insight derived from this approach. Additional MeT Channels in Animals The TRP channel genes are conserved in and encode proteins complications include alteration of channel function by their predicted to have six transmembrane domains and assemble into tetrameric association with heteromeric channel complexes and through ion channels. Many TRPs have ankyrin repeats in their intracellular amino alternative splicing of ion channel genes. terminal; some have more than ten such repeats (Venkatachalam and Montell, 2007). The DEG/ENaC genes are absent from plants, yeast, and other microbes, but conserved in animals (Goodman and Schwarz, 2003). They Stagecraft for Sensory Biology encode proteins with two transmembrane domains and a large extracellular A fundamental block to progress in understanding how mecha- domain. Three DEG/ENaC proteins assemble to form an ion channel. Both noreceptor neurons function is that studying stimulus-initiated TRP and DEG/ENaC proteins can form homomeric and heteromeric channels, increasing the potential for channel diversity. Recently, two addi- behavior, action potential generation, or intracellular calcium tional classes of membrane proteins (Piezo and TMC) have been linked to dynamics does not allow researchers to separate the initial step in (Coste et al., 2010; Kawashima et al., 2011) of mechanotransduction from amplification, gain control, and and Drosophila fruitflies (Kim et al., 2012). Piezo is sufficient to produce stretch activated channels in heterologous cells (Coste et al., 2010, 2012; Bae et al., transmission. Behavior, spikes, and calcium dynamics depend 2011) and purified Piezo forms a channel in lipid bilayers (Coste et al., 2012). on the action of all of the ion channels expressed by sensory TMC1 and TMC2 are required for mechanotransduction by - and neurons. These channels are interconnected in feedback loops vibration-sensing cells in mice (Kawashima et al., 2011). Both Piezo and regulated by a common control variable—. TMC have homologs in invertebrates; a C. elegans TMC homolog is expressed in the multidendritic PVD nociceptors. The predicted topology and stoichi- The voltage-clamp uncouples such feedback loops by holding ometry of Piezo and TMC await further experimental confirmation. membrane potential constant and allows researchers to examine transduction independently of amplification, gain control, and proteins enriched in mechanoreceptor neurons and required for spike generation. Within this heuristic, deleting molecules their function. Figure 1 summarizes current knowledge about needed for the formation or function of MeT channels should elim- proteins proposed to form MeT channels in animals. The mec- inate mechanoreceptor currents but leave other ionic currents 4 DEG/ENaC and osm-9 TRP channel genes were the first candi- and mechanisms for amplification, gain control and spike gener- dates identified from classical genetic screens. The DEG/ENaC ation intact. Conversely, deleting molecules essential for post- genes are conserved in animals, but absent from plants, transduction signal should leave mechanoreceptor currents bacteria, and fungi (Goodman and Schwarz, 2003; Hunter intact but produce defects in other ionic currents or in amplifica- et al., 2012) and encode proteins with two transmembrane tion, gain control, and spike generation. Marrying in vivo voltage domains and a large extracellular domain. As revealed in high- clamp with genetic dissection of identified mechanoreceptor resolution crystal structures (Gonzales et al., 2009; Jasti et al., neurons in C. elegans has revealed that the pore-forming subunits 2007), three DEG/ENaC proteins assemble to form an ion of MeT channels are DEG/ENaCs in two classes of mechanore- channel. Both homomeric and heteromeric channels have ceptors (Geffeney et al., 2011; O’Hagan et al., 2005) and a TRP been observed (Akopian et al., 2000; Deval et al., 2004; Donier channel operates in a third class (Kang et al., 2010). et al., 2008; Gru¨ nder et al., 2000; Hesselager et al., 2004; Lingue- glia et al., 1997). The TRP channel genes comprise a large super- Neuron by Neuron family conserved in eukaryotes and encode proteins predicted C. elegans nematodes are microscopic animals with a compact to have six transmembrane domains. Four TRP channel proteins nervous system consisting of only 302 neurons, about 30 of

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which are classified as mechanoreceptor neurons. Because the From analysis of ASH, we learn that DEG/ENaC channels can mechanoreceptor neurons can be identified in living animals, act in parallel with a second MeT channel and that TRPV chan- and because of their small size, it is possible to record mechano- nels are important for posttransduction signaling. This complex receptor currents (MRCs) and mechanoreceptor potentials pathway for mechanoreceptor neuron signaling may be shared (MRPs) in vivo. MRCs have been recorded from the body touch with other nociceptors responsible for detecting noxious and receptor neurons known collectively as the TRNs, the cephalic potentially damaging sensory stimuli. An additional, conserved CEP neurons and two classes of nociceptors, the ciliated ASH function of nociceptors is their sensitization in response to injury neurons and the multidendritic PVD neurons. In all four of these and their regulation by biogenic amines (Walters and Moroz, mechanoreceptors, stimulation activates inward currents 2009). Such sensitization is also apparent in C. elegans and re- (Figure 3) and evokes transient increases in intracellular calcium. flected in the finding that ASH-dependent behaviors are regu- Strikingly, MRCs are activated in response to both the applica- lated by various biogenic amines, including serotonin (Chao tion and withdrawal of stimulation. Such response dynamics et al., 2004). Collectively, these observations raise the possibility were first described 50 years ago in recordings from Pacinian that biogenic amines might regulate the sensitivity of nocicep- corpuscles in mammals (Alvarez-Buylla and Ramirez De Are- tors to mechanical cues and that such regulation may affect llano, 1953; Gray and Sato, 1953) and are emerging as MeT channels, posttransduction signaling or both. a conserved property of somatosensory mechanoreceptor The multidendritic PVD neuron is a polymodal neuron acti- neurons. vated by mechanical and thermal stimuli and is proposed to The TRNs (ALM, PLM, AVM, and PVM) express several DEG/ function as a nociceptor. Like ASH, PVD expresses multiple ENaC channel proteins, but no TRP channel subunits have been TRP and DEG/ENaC channel subunits (Figure 2A). As in ASH reported (Figure 2A). External mechanical loads open sodium- and the touch receptor neurons, mechanoreceptor currents in dependent, amiloride-sensitive mechanotransduction (MeT) PVD are amiloride sensitive and sodium dependent (Li et al., channels. MEC-4 is essential, while MEC-10 is dispensable 2011b). These biophysical properties suggest that MRCs are for the generation of MeT currents (Arnado´ ttir et al., 2011; O’Ha- carried primarily by a DEG/ENaC channel. Consistent with this gan et al., 2005). Both proteins are pore-forming subunits of the idea, Chatzigeorgiou et al. (2010) demonstrated that both native MeT channel since missense mutations of a conserved DEGT-1 and MEC-10 are required for mechanically evoked glycine in the second transmembrane domain alter the perme- calcium transients in PVD. But, as reported for the touch ability of the MeT current (O’Hagan et al., 2005). These protein receptor neurons (Arnado´ ttir et al., 2011), loss of mec-10 had partners were the first to be linked to native MeT currents in any no effect on mechanoreceptor currents (Li et al., 2011b). Thus, animal. mec-10 may function redundantly with other DEG/ENaC chan- CEP expresses at least one DEG/ENaC and one TRP channel nels expressed in PVD, including degt-1, del-1, asic-1. Additional protein, TRP-4 (Figure 2A). Two lines of evidence support the studies are needed to clarify this issue and to determine the func- idea that the TRP-4 protein is an essential pore-forming subunit tion of all of these ion channel proteins in PVD. One approach of MeT channels in CEP: (1) loss of TRP-4 eliminates MRCs in developed recently exploits optogenetics to identify PVD-ex- CEP and (2) mutations in the putative pore domain of the channel pressed genes needed for posttransduction signaling (Husson alter the reversal potential of MRCs (Kang et al., 2010). These et al., 2012). Using this strategy, Husson et al. (2012) show that latter data are strong indicators that TRP-4 is a pore-forming PVD-selective knockdown of asic-1 alters light-evoked behav- subunit of the MeT channel in CEP. ioral responses. (Light responses were unaffected in animals The ASH neurons function as nociceptors in the animal by mec-10, del-1, and degt-1 knockdown.) because they require more intense forces for activation than PVD appears to express seven TRP channels, including PLM and larger displacements for activation than CEP (Geffeney TRPA-1 and OSM-9 (Figure 2A). Neither TRPA-1 nor OSM-9 et al., 2011). These cells express multiple DEG/ENaC and TRP are required for calcium transients induced by noxious mechan- channel proteins (Figure 2A), but the major mechanoreceptor ical stimuli. However, TRPA-1 is needed for responses to current is carried by a MeT channel formed by the DEG/ENaC noxious cold (Chatzigeorgiou et al., 2010). Less is known about channel protein, DEG-1. A minor current remains in deg-1 null the function of the other TRP channels, but an optogenetics- mutants and is carried by a biophysically distinct channel based approach reveals that GTL-1 is required for normal (Geffeney et al., 2011). Though it is possible that DEG-1 and light-evoked behaviors and strongly suggests that this TRPM the channel responsible for the minor current function in series channel plays an essential role in posttransduction signal ampli- with DEG-1 amplifying the minor current, the data support fication (Husson et al., 2012). Collectively, these studies paint a model where the channels function in parallel because loss a picture of PVD function in which a DEG/ENaC channel acts of DEG-1 does not alter the rise rate of MRCs in ASH. The as a force sensor, TRPA-1 detects thermal stimuli, and ASIC-1 TRPV proteins OSM-9 and OCR-2 are essential for ASH- and GTL-1 contribute to posttransduction signaling. mediated behaviors (Colbert et al., 1997; Tobin et al., 2002), The FLP neurons are multidendritic neurons and are activated but loss of these channel subunits has no effect on either the by noxious mechanical and thermal stimuli (Chatzigeorgiou and major or minor current in ASH (Geffeney et al., 2011). In ASH, Schafer, 2011; Chatzigeorgiou et al., 2010). The FLP neurons TRPV channels likely regulate activity downstream of innervate the body surface anterior to PVD and co-express mechanotransduction, as suggested by their importance for osm-9 and three DEG/ENaC genes: mec-10, unc-8, and del-1 calcium signaling in ASH following mechanical stimulation (Figure 2A). Apart from the observation that mechanical stimuli (Hilliard et al., 2005). activate calcium transients in FLP in a MEC-10-dependent

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Figure 2. DEG/ENaC and TRP Channel Proteins Coexpressed in Mechanoreceptor Neurons in C. elegans Nematodes, Drosophila melanogaster Fruitflies, and Mammals, including Mus musculus Mice This graphical table illustrates the gross morphology of entire mechanoreceptor neurons (C. elegans) or peripheral sensory endings (Drosophila, mice and other mammals) and lists ion channel subunits that are expressed in each class of mechanoreceptor cell. Sources for C. elegans mechanoreceptor expression are listed by number above: (1) Driscoll and Chalfie, 1991; (2) Huang and Chalfie, 1994; (3) Chatzigeorgiou et al., 2010; (4) Smith et al., 2010;(5)Colbert et al., 1997; (6) Kindt et al., 2007; (7) Tavernarakis et al., 1997; (8) Hall et al., 1997;(9)Tobin et al., 2002; (10) Voglis and Tavernarakis, 2008; (11) Walker et al., 2000; (12) Li et al., 2006. Drosophila melanogaster sources are as follows: (13) Gong et al., 2004; (14) Kim et al., 2003; (15) Lee et al., 2010; (16) Liang et al., 2011; (17) Gallio et al., 2011; (18) Lee et al., 2005; (19) Liu et al., 2007; (20) Hamada et al., 2008; (21) Kim et al., 2010; (22) Cheng et al., 2010; (23) Chen et al., 2010; (24) Liu et al.,

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TRP TRP DEG/ENaC These examples in C. elegans nematodes establish the rule CEP PDE PVD that mechanoreceptor neurons commonly express multiple I I I DEG/ENaC and TRP channel proteins and that these channels

z z z operate together to enable proper sensory function. The ability [Kang et al 2010] [Li et al 2011] [Li et al 2011] to directly measure MRCs in vivo has revealed that both DEG/ENaC and TRP channels can form MeT channels. Evidence from the ASH and PVD nociceptors suggests that some TRP channels are essential for posttransduction events needed for sensory signaling. These case studies provide evidence for the ASH F idea that TRP channels can be crucial elements in both sensory I transduction and in post-transduction signaling. They also illus- F I trate the powerful insights available when detailed physiological [Geffeney et al 2011] analysis of identified mechanoreceptor neurons is merged with DEG/ENaC [O’Hagan et al 2005] genetic dissection. DEG/ENaC MRPs in Pacinian Corpuscles It is rare for deletion of a single DEG/ENaC gene to induce z strong behavioral defects in C. elegans. Indeed, there is only V one such DEG/ENaC gene known so far: mec-4. By contrast, [Mendelsson 1964] deleting the DEG/ENaC genes mec-10, deg-1, unc-8, and unc-105 fails to produce clear behavioral phenotypes, although Figure 3. Mechanoreceptor Currents in C. elegans gain-of-function alleles significantly disrupt several behaviors. Mechanoreceptor Neurons Activate in Response to the Application and Removal of Mechanical Stimulation Though only a subset of the DEG/ENaC genes have been studied Mechanoreceptor currents have been recorded using in vivo whole-cell patch- in this way, these findings suggest there is considerable redun- clamp recording and the predominant ion channel type identified by genetic dancy in C. elegans . The case of mec-4 dissection. Traces adapted from: PLM, O’Hagan et al. (2005); CEP, Kang et al. and mec-10 illustrate this idea clearly: both genes are coex- (2010); PDE and PVD, Li et al. (2011b); ASH, Geffeney et al. (2011). Displacement stimuli were applied to activate CEP, PDE, and PVD, while pressed in the TRNs and encode pore-forming subunits of the mechanical stimuli delivering known forces were applied to activate PLM and MeT channel required for gentle touch sensation (O’Hagan ASH. Receptor potentials in PLM and ASH mirror the receptor currents (Gef- et al., 2005). Whereas deleting mec-4 eliminates mechanore- feney et al., 2011; O’Hagan et al., 2005) and are reminiscent of the response dynamics of Pacinian corpuscles in cats (inset, lower left). ceptor currents and behavioral responses to touch, deleting mec-10 produces a mild defect in touch sensation and has little effect on mechanoreceptor currents (Arnado´ ttir et al., 2011). manner, little is known about the function of MEC-10, UNC-8, and DEL-1 in FLP. The contribution of TRP channel genes to Case Studies on the FLP function is complex, as FLP mechanosensitivity depends The peripheral nervous system of Drosophila larvae has three on OSM-9 and TRPA-1-dependent signaling in the OLQ mecha- main types of neurons (Bodmer et al., 1987; Bodmer and Jan, noreceptors (Chatzigeorgiou and Schafer, 2011). 1987; Ghysen et al., 1986). External sensory and chordotonal In a cell that expresses multiple TRP channel subunits and no neurons have a single sensory dendrite and innervate specific DEG/ENaC subunits, each TRP protein appears to have a distinct mechanosensory organs. In contrast, multidendritic neurons cellular function. The OLQ neuron expresses three TRP channel have a variable number of fine dendritic processes that lie subunits and two of these have been examined for their role in beneath the epidermis and do not innervate a specific structure. initiating behavioral responses and calcium transients in Different subclasses of these neurons provide information about response to mechanical stimulation. Loss of TRPA-1 decreases touch and body position as well as function as nociceptors two behaviors influenced by OLQ, the cessation of foraging for (Hughes and Thomas, 2007; Song et al., 2007; Zhong et al., food and reversal of forward movement induced by mechanical 2010). In the adult, external sensory and chordotonal neurons stimulation (Kindt et al., 2007). Surprisingly, loss of TRPA1 had innervate more elaborate structures formed by the cuticle a very subtle effect on calcium transients in OLQ. The first including bristles and antennae. These cells continue to be response to mechanical stimulus was not affected, but the responsible for and touch sensation. Multiden- magnitude of the second response was reduced (Kindt et al., dritic neurons persist into adulthood after extensive arbor rear- 2007). A role for the TRPV channel subunit OSM-9 is evident rangements (Shimono et al., 2009). from the finding that osm-9 mutant OLQ neurons lack mechani- Polymodal nociceptor neurons in Drosophila larvae called cally-evoked calcium transients (Chatzigeorgiou et al., 2010). class IV multidendritic or md neurons innervate the body surface Because MRCs have yet to be measured in this mechanore- and express both TRP and DEG/ENaC channel subunits ceptor neuron, it is not known whether loss of TRPA-1 or (Figure 2B). These neurons initiate aversive, nocifensive OSM-9 affect MRCs or the events that follow their activation. responses to heat, mechanical loads and UV light (Hwang

2003; (25) Bechstedt et al., 2010; (26) Tracey et al., 2003; (27) Zhong et al., 2012; (28) Adams et al., 1998; (29) Zhong et al., 2010. These sources establishing expression in peripheral endings in mice and humans were consulted: (30) Calavia et al., 2010; (31) Montan˜ o et al., 2009; (32) Drummond et al., 2000; (33) Garcı´a-An˜ overos et al., 2001;(34)Price et al., 2001; (35) Suzuki et al., 2003b; (36) Kwan et al., 2009; (37) Price et al., 2000;(38)Fricke et al., 2000; (39) Xu et al., 2002.

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et al., 2012; Tracey et al., 2003; Xiang et al., 2010; Zhong et al., (NOMPC), and that the TRPV proteins Nan and Iav are expressed 2010, 2012). The md neurons express three TRPA genes: broadly and needed for both and gravity sensing. painless, pyrexia and dTRPA1 (Figure 2B). None of these are ex- In sound-sensitive chordotonal neurons, the exact function of pressed exclusively in md neurons, suggesting that they have each TRP channel is matter of continuing investigation. One additional functions. Painless is present in the larval cardiac model (Go¨ pfert et al., 2006) is that NOMPC is essential for de- tube (Se´ natore et al., 2010) and in adult sensilla, including gusta- tecting sound-induced mechanical stimuli and Nan and Iav tory bristles in the proboscis, the leg and the wing margin (Al-Anzi work together to both refine mechanical amplification and et al., 2006); Pyrexia is expressed in neurons that innervate ensure the proper transmission of stimulus-evoked action sensory bristles and antennae (Lee et al., 2005); and dTRPA1 potentials in the antennal . In this schema, NOMPC func- is expressed both in neurons and in central tions like its C. elegans homolog, TRP-4, and forms the pore of neurons required for temperature-sensing in adult flies (Hamada a sensory MeT channel. Techniques for measuring mechanore- et al., 2008; Kim et al., 2010). ceptor currents in the JO are needed to directly test this model, The contribution of Pyrexia to the mechanosensitivity of md but functional specialization of NOMPC and Nan/Iav is sup- neurons has not been studied, but genetic deletion of Painless ported by the fact that they occupy distinct compartments in and dTRPA1 increase the threshold for aversive responses to the sensory of JO mechanoreceptors (Lee et al., 2010; heat and force (Tracey et al., 2003; Zhong et al., 2012). In contrast, Liang et al., 2011). loss of either a DEG/ENaC channel subunit, Pickpocket, or Several TRP proteins may be coexpressed in the chordotonal DmPiezo reduces the response to intense mechanical stimuli, organs of the adult leg that provide information about joint posi- but has no effect on the response to noxious heat (Kim et al., tion (Gong et al., 2004; Kim et al., 2003; Liang et al., 2011). These 2012; Zhong et al., 2010). Decreasing the expression of both include NOMPC, Iav, and Waterwitch (Wtrw), which appear to be Pickpocket and DmPiezo renders larvae insensitive to noxious coexpressed in the campaniform sensilla that detect cuticle mechanical stimuli, but has little effect on responses to noxious deformation in the wings and (Gong et al., 2004; Kim heat. Additionally, cultured md neurons from DmPiezo knockout et al., 2003; Liang et al., 2011). The coexpression of these proteins mutants lack mechanically activated currents that are present in in other mechanoreceptor neurons suggests that an under- cell isolated from wild-type animals (Kim et al., 2012). These find- standing of how these cells enable mechanosensitivity may ings suggest that Pickpocket and DmPiezo could function in depend on cellular context and the entire ensemble of ion chan- parallel as subunits of MeT channels in md neurons. nels expressed in each mechanoreceptor. Finally, NOMPC is Recent studies reveal that the painless and dTRPA1 genes famous for its expression in the mechanoreceptors that innervate encode multiple isoforms (Hwang et al., 2012; Zhong et al., large bristle sensilla on the fly’s body (Walker et al., 2000). NompC 2012). The longest isoform of Painless, Painlessp103, has eight mutants lack transient, but retain sustained trans-epithelial mech- ankyrin repeats in the amino-terminal domain and the shortest, anoreceptor currents (Walker et al., 2000). Thus, in bristle mech- Painlessp60, has none. Both isoforms are expressed in md anoreceptors, there appears to be another mechanotransduction neurons, but only the shortest isoform rescues mechanonoci- channel. Bristle receptors express many other TRP channel ception (Hwang et al., 2012). In contrast, dTRPA1 isoforms differ subunits as well as DEG/ENaC channel subunits (Figure 2B). in regions of the protein that flank the ankyrin repeats (Zhong These data raise the possibility that another channel may function et al., 2012) and two isoforms of the gene are expressed in md in parallel to the NOMPC channel in the bristle receptor neuron. neurons. One isoform, dTrpA1-C, restores normal thermal noci- ception but not mechanonociception. It is not clear whether The Company of and Nematodes dTRPA1-D or an as-yet-unidentified isoform is important for me- There is evidence that DEG/ENaC and TRP channels function as chanonociception in md neurons. Thus, an unexpectedly MeT channel subunits in distinct mechanoreceptor neurons in complex model for md neuron-mediated mechanonociception both C. elegans and Drosophila. One evolving paradigm is that is emerging—Pickpocket and DmPiezo detect mechanical loads mechanonociceptors (md neurons in Drosophila and the PVD in parallel, while Painlessp60 and perhaps a dTRPA1 isoform are and ASH neurons in C. elegans) rely on DEG/ENaC proteins to required for post-transduction signaling, including amplification. detect noxious mechanical stimuli and on TRP channels for The Johnston’s organ (JO) of adult Drosophila antennae is essential post-mechanotransduction signaling. Another is the a near-field sound receptor and like other animal , the JO notion that TRP channels can play multiple roles within a single relies on mechanical amplification and frequency-selective mechanoreceptor neuron, exemplified by the finding that a trio tuning to optimize sound sensitivity (Go¨ pfert et al., 2005, 2006; of TRP channels is critical for mechanotransduction and post- Robert and Go¨ pfert, 2002; Tsujiuchi et al., 2007). Sound is not transduction signal essential for hearing in Drosophila. A third the only mechanical stimulus detected by the JO, however. paradigm is the presence of multiple MeT channels as found in This array of hundreds of mechanoreceptor neurons also Drosophila bristles, md neurons and C. elegans ASH nocicep- responds to displacements induced by wind and gravity tors, suggesting that functional redundancy may be a shared (Kamikouchi et al., 2009; Sun et al., 2009; Yorozu et al., 2009). feature of mechanoreceptors. Mechanoreceptor cells in the JO project their into the antennal nerve and express five TRP channels (Figure 2B): The Mouse and Its Skin NOMPC, Nan, Iav, Painless, and Pyrexia. Genetic dissection of As in nematodes and flies, mechanoreceptor neurons in mice hearing and gravitaxis reveals that some channels (Painless, coexpress DEG/ENaC and TRP channel proteins and are among Pyrexia) are needed to gravity, others for hearing the principal actors that give rise to somatic sensations. Except

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Figure 4. Classification Schemes for Skin Mechanoreceptors in Mammals Mammalian mechanoreceptor nerve fibers can be classified according to three physiological prop- erties: (1) the speed of action potential propagation (values shown here are for humans), (2) the threshold for activation, and (3) the rate of adap- tation to mechanical stimuli. The categories of Ab, Ad, and C fibers are defined by their propagation speeds where Ab-fibers have the most rapid propagation speeds and the slender, unmyelin- ated C fibers have the slowest. Most fibers in these categories share other properties. For example, C fibers have slow rates of adaptation to mechanical stimuli and many have high mechanical thresh- olds. In contrast, most Ab fibers have low mechanical thresholds and these fibers are thought to innervate light-touch receptors in the skin.

for nociceptors associated with painful perceptions, the perfor- Genetic deletion of single DEG/ENaC or TRP channel proteins mance of mechanoreceptors in mammals depends on affiliation in mice alters sensitivity to mechanical stimulation, but leaves with specialized sensory organs in the skin presumed to be the both functions largely intact. While these studies cast doubt on locus of mechanotransduction. Figure 2C summarizes current the idea that DEG/ENaC or TRP channel proteins are essential research showing that DEG/ENaC and TRP channel proteins for mechanotransduction in mammals, they also suggest that localize to the skin in mice, a property that suggests these the mammalian is robust to genetic dele- proteins could form MeT channels in mammals. While this review tion. Such robustness could reflect molecular redundancy within focuses on sensory neurons, skin epithelial cells, including or between ion channel gene families. Additionally, robustness Merkel cells, also play important roles in mechanosensation could be conferred by functional degeneracy among mechano- (see Lumpkin and Caterina, 2007). Most investigations of receptor neurons. The potential for degeneracy arises from the mechanotransduction in mammals have relied on behavioral fact that skin dermatomes contain a mixture of peripheral studies, analysis of dorsal root ganglia (DRG) or trigeminal (TG) sensory structures and are innervated by multiple classes of neurons in culture, and extracellular, single-unit recordings in vivo somatosensory neurons. For example, low-threshold, rapidly and ex vivo. The recent demonstration of in vivo, whole-cell adapting Ab fibers are thought to innervate both Pacinian and patch-clamp recordings from DRG neurons (Ma et al., 2010)is Meissner corpuscles in the skin (Brown and Iggo, 1967; Burgess an exciting new tool that is just beginning to be applied. et al., 1968; Vallbo et al., 1995). In addition to having distinct The nerve endings emanating from TG and DRG neurons are morphologies, each of these endings also expresses different diverse and are classified according to the expression of DEG/ENaC and TRP channel proteins (Calavia et al., 2010; Gar- signaling peptides and receptors, their functional properties or cı´a-An˜ overos et al., 2001; Kwan et al., 2009; Price et al., 2001; their morphology and (see Figure 4 and Delmas et al., Suzuki et al., 2003b). In this scenario, loss of a single ion channel 2011; Lewin and Moshourab, 2004). For most somatosensory protein is expected to have only a minor effect on the entire class neurons, however, anatomical and functional properties are of such fibers. only loosely connected (Boulais and Misery, 2008). For instance, The acid-sensing ion channels or ASICs are a vertebrate sub- fibers that share multiple electrophysiological features such as division of the conserved DEG/ENaC superfamily. Most if not all Ab-like conduction velocities as well as rapidly-adapting, low- of the ASIC proteins are expressed in cell bodies in the trigeminal threshold responses to mechanical stimuli innervate multiple and dorsal root ganglia (reviewed in Deval et al., 2010) and peripheral end organs (Brown and Iggo, 1967; Vallbo et al., localize to the peripheral endings in the skin (Figure 2C). For 1995). An example of the converse situation in which fibers instance, ASIC1 is expressed in innervating Pacinian with distinct electrophysical properties innervate a common corpuscles in (Calavia et al., 2010; Montan˜ o et al., peripheral end organ has recently come to light: Li et al. 2009). However, genetic deletion of ASIC1 has no effect on the (2011a) show that Ab,Ad, and C fibers all form lanceolate threshold or firing frequency of fibers innervating mouse skin endings that surround hair follicles. This discovery relied on (Page et al., 2004), but alters visceral sensory function (Page developing a suite of genetic markers that were exploited in et al., 2004; Page et al., 2005). ASIC2 and ASIC3 are expressed two ways. First, they were used to determine the peripheral in the majority of mechanoreceptor endings in mouse skin endings associated with marked sensory subtypes. Second, (Figure 2C; Garcı´a-An˜ overos et al., 2001; Price et al., 2000, they were used to link electrophysiological properties derived 2001), but deleting ASIC2 or ASIC3 has only subtle effects on from in vivo intracellular recordings to the marker suite and the activity of mechanoreceptor fibers (Price et al., 2000, hence to mechanoreceptor subtype. Thus, knowledge of 2001). Moreover, neither ASIC2 nor ASIC3 is essential for MeT conduction velocity and force sensitivity may not be sufficient currents studied in cultured DRG neurons (Drew et al., 2004). to infer the identity of the peripheral organ being stimulated. Collectively, these investigations indicate that no single ASIC

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subunit is essential for the function of mechanoreceptor neurons Two others have recently joined their ranks: Piezo and TMC. in mice and suggest that the function of such neurons is robust to Here, we surveyed the literature to establish that most, if not all genetic deletion of channel proteins. mechanoreceptor neurons in worms, flies, and mice express In principle, genetic deletion of a shared auxiliary subunit multiple DEG/ENaC and TRP channel proteins. Piezo is ex- could reveal more severe deficits in behavioral and cellular pressed in a subset of somatosensory neurons in mice, but its responses to touch because such proteins might affect the func- representation relative to other channels is not known. Little is tion of multiple channel-forming subunits. The impact of genetic known about expression of TMC proteins in mechanoreceptor deletion of SLP3 illustrates the power of this idea (Wetzel et al., neurons. But, the landscape of ion channel coexpression in mech- 2007). SLP3 (also known as STOML3) is a stomatin-like protein anoreceptor neurons is only beginning to be mapped. Future work that binds to both ASIC2 and ASIC3 and alters the activity of aimed at refining such maps for mammalian mechanoreceptor ASIC channels in heterologous cells. It is orthologous to the neurons will be critical for deeper understanding. Also, each of C. elegans protein MEC-2, which is required for MeT currents these potential MeT channel subunits operates within a large in vivo and enhances MEC-4-dependent currents in heterolo- company of other ion channel actors that increase the complexity, gous cells (Goodman et al., 2002; Huang and Chalfie, 1994; flexibility, and robustness of somatosensory neuron function. O’Hagan et al., 2005). Genetic deletion of SLP3 decreases the Both DEG/ENaC and TRP channel proteins can function as proportion of mechanically sensitive Ab and Ad fibers that inner- essential, pore-forming subunits of MeT channels in three vate the skin and the proportion of dissociated DRG neurons classes of mechanoreceptor neurons in worms: the touch with mechanosensitive currents (Wetzel et al., 2007). Addition- receptor neurons, the CEP texture sensors, and the ASH noci- ally, loss of SLP3 disrupts texture sensing. These data suggest ceptors. Unexpectedly, some mechanoreceptor neurons rely that many, but not all mechanoreceptors depend on SLP3 and on a single class of channels to detect mechanical cues (TRNs its DEG/ENaC binding partners to detect mechanical stimuli. and CEP), while others (ASH) use at least two, genetically and bi- Mirroring the effects of single ASIC gene deletions are those of ophysically distinct channels. Although such functional redun- TRP channel gene deletions: loss of a single channel gene has dancy has been established only in invertebrates so far, it could only subtle effects on somatosensory nerve fiber function and explain some of the notable failures of genetic deletion of puta- no single TRP channel gene deletion leads to a loss of mechano- tive MeT channel subunits to disrupt touch and pain sensation sensitivity in an individual fiber class (Kwan et al., 2006, 2009; in mice. From worms we also learn that some, but not all pore- Liedtke and Friedman, 2003; Suzuki et al., 2003a). But, loss of forming MeT channel subunits are essential for mechanosensa- TRP channel proteins has clear effects on the response of noci- tion. This situation is likely to exist in other mechanoreceptor ceptors to inflammation. Here, we provide three examples. First, neurons, including those responsible for touch and pain noxious chemical agents such as mustard oil potentiate behav- sensation in mammals. These findings recommend adopting ioral responses to mechanical stimuli, an effect which is muted in a cautionary stance in interpreting the modest effects of genetic TRPA1 knockout mice (Bautista et al., 2006). Loss of TRPA1 also deletion of a single putative MeT channel subunit. disrupts sensitization produced by injection of bradykinin, a peptide released by damaged tissue (Kwan et al., 2009). Epilogue Second, genetic deletion of TRPV4 has subtle effects on behav- Somatic sensation of gentle and noxious mechanical cues gives ioral and neural responses to mechanical cues (Chen et al., 2007; rise to our sense of touch and acute pain and also provides Suzuki et al., 2003a) but produces significant deficits in inflam- crucial information that regulates body movements and essential mation-induced sensitization. Compounds induced by inflam- functions like . The robustness that makes full mation (prostaglandin E2 and serotonin) decrease the threshold loss of somatosensory function extremely rare complicates for mechanical activation of C fiber nociceptors in wild-type, but traditional genetic dissection of somatic sensation in mammals. À/À not in TRPV4 (Alessandri-Haber et al., 2005; Chen et al., Progress toward identifying the composition of MeT channels in 2007). In addition, the spontaneous activity of C fibers is mammalian mechanoreceptor neurons would be enhanced by increased following inflammation, but there is no change in the refining current methods for categorizing somatosensory À/À rate of spontaneous activity in C fibers isolated from TRPV4 neurons and their fibers to better reflect their functional organiza- mice. Finally, TRPC1-deficient mice have reduced firing rates tion. Perhaps, following the example of Li et al. (2011a) and b in A -fibers and reduced probability of withdrawal from light mapping the channel proteins coexpressed in peripheral touch (Garrison et al., 2012). A reduction of TRPC1 has a more endings in the skin may provide a reliable method for linking dramatic effect on the ability of the animals to respond to the morphological subtypes to specific neuronal functions. When inflammation mediators, prostaglandin E2 and serotonin (Ales- robust categorization of DRG and TG neurons can be combined sandri-Haber et al., 2009). Thus, loss of TRP channels has subtle with subtype-selective gene markers, the curtain could rise on effects on baseline responses and significant effects on the a new scene in which selected classes of sensory neurons can ability of animals to respond to inflammation. be identified and targeted for in vivo whole-cell patch recording in transgenic mice, as they are in worms. Conclusions and Perspectives Over the past decades, a great deal of attention has focused on ACKNOWLEDGMENTS discovering the protein partners that form MeT channels in somatic mechanoreceptors. Two classes of ion channel proteins We thank the Goodman laboratory for lively discussion; three anonymous are leading candidates: DEG/ENaC and TRP channel proteins. reviewers; Rebecca Agin for artwork contributed to Figures 1 and 2; and we

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